Sterol composition of isolates of Erysiphe graminis f.sp. tritici differing in sensitivity to fenpropimorph

Isolates of Erysiphe graminis f.sp. tritici with wild-type or reduced sensitivity to fenpropimorph were similar in sterol composition, viz. ergosta-5,24(28)-dienol (±90%) and episterol (±10%). Following treatment with fenpropimorph, the relative content of episterol increased in conidia of all isolates tested, while that of ergosta-5,24(28)-dienol decreased. These results suggest that fenpropimorph, under the test conditions used, does not inhibit activity of sterol Δ¹⁴-reductase or Δ⁸→Δ⁷-isomerase but probably interferes with the final part of the demethyl sterol synthesis. However, modifications in this part of the pathway are probably not responsible for the decreased sensitivity of the pathogen to the fungicide. © 1998 SCI.     

Specific effects of tridemorph on sterol biosynthesis in Ustilago maydis

In an attempt to indicate the site of action of tridemorph in ergosterol biosynthesis by Ustilago maydis the nature of the sterol intermediates accumulating in treated cells was studied. At low growth-inhibiting concentrations of the toxicant (10 and 15 μg/ml) decline of ergosterol content during 6 hr of incubation was accompanied by an accumulation of various sterol intermediates of which ergosta-8,14-dien-3β-ol appeared to be the major sterol. After isolation of this intermediate its identity was further confirmed by direct comparisonof its ultraviolet, glc, and mass spectrum with those of an authentic synthesized sample of ergosta-8,14-dien-3β-ol (ignosterol). Results indicate that toxicity of tridemorph is caused by a specific inhibitive effect on the enzyme Δ¹⁴-reductase which is responsible for $\text{14}\text{15}$ double-bond reduction in sterol biosynthesis.     

Conformational analysis of the fungicide fenpropimorph by molecular mechanics calculations and NMR spectroscopy

Fenpropimorph is an inhibitor of Δ8→Δ7-isomerase and Δ14-reductase in fungi and cycloeucalenol-obtusifoliol-isomerase and Δ8→Δ7-isomerase in higher plants. A detailed conformational analysis of the most potent enantiomer (S)-fenpropimorph is described. The conformational analysis is based on three different methods: molecular mechanics calculations. NMR spectroscopy and X-ray crystallography. In solution several conformations coexist. The molecular mechanics calculations using MM2(85) show a folded conformation of the unprotonated form of fenpropimorph to be stabilized by at least 3 kcal mol^?1 relative to the next lowest energy conformer, suggesting only one conformation of fenpropimorph to be present. A corresponding stabilization of the protonated form (5.4 kcal mol^?1) is obtained. However, this stabilization is found partly to be due to attractive steric interactions and may be a phenomenon occurring only in vacuum. In the solid state, fenpropimorph exists in an L-shaped conformation resembling one of the low-energy conformers obtained with MM2(85). Based on comparison with the natural substrate, a totally extended conformation of fenpropimorph is suggested to be the biologically active conformation for interaction with the Δ8 → Δ7-isomerase in fungi.     

Effects of triazole inhibitors of sterol biosynthesis on the free and esterified sterol composition of celery cell suspension cultures

Treatment of celery cell suspension cultures with paclobutrazol and three other triazoles resulted in decreased growth and an accumulation of 14α-methylsterols in both the free sterol and the steryl ester pools, thus indicating that the triazoles were inhibiting the action of the plant obtusifoliol 14α-demethylase system. Obtusifoliol, 14α-methylcampesta-8,24(24¹)-dien-3β-ol and 14α-methylcampest-8-en-3β-ol were the main 14α-methylsterols to increase in the free sterol pool. In the presence of the triazoles the steryl esters became virtually depleted of sitosterol, campesterol, stigmasterol and isofucosterol, which were replaced by 14α-methylsterols with obtusifoliol becoming the dominant esterified sterol. Treatment of the celery culture with a fourth triazole which did not have a noticeable effect on growth and caused negligible accumulation of 14α-methylsterols in the free sterol pool nevertheless produced some enrichment of the steryl esters in obtusifoliol. The results indicate that, following triazole treatment, a rapid esterification of the accumulating 14α-methylsterol intermediates occurs before they start to build up in significant amount in the free sterol pool.     

Effects of sterol biosynthesis inhibitor fungicides in the phytopathogenic fungus,Nectria haematococca: ergosterol depletion versus precursor or abnormal sterol accumulation as the mechanism of fungitoxicity

The relative importance of the depletion of ergosterol versus the accumulation of precursor or abnormal sterols in the mechanism of fungal growth inhibition by sterol biosynthesis inhibitor fungicides is incompletely understood. In order to investigate this problem further, the degree of inhibition of the growth of Nectria haematococca by fungicides with different enzymatic targets in the sterol biosynthetic pathway was determined and compared with their effects on the sterol profile. The sensitivity of N. haematococca was highest towards fenpropimorph, followed by tebuconazole, terbinafine, fenpropidin and tridemorph. Terbinafine, a squalene epoxidase inhibitor, induced a very large accumulation of squalene without very significant inhibition of ergosterol biosynthesis and growth. The fungus appeared able to tolerate large amounts of squalene. In the case of tebuconazole, a sterol 14α-demethylase inhibitor, it seemed that the accumulation of C4 mono- and dimethyl sterols was responsible for fungitoxicity. Fenpropimorph and fenpropidin seemed to be good inhibitors of both sterol Δ¹⁴-reductase and Δ⁸→Δ⁷-isomerase, whereas tridemorph was a better inhibitor of Δ⁸→Δ⁷-isomerase than of the Δ¹⁴-reductase. Large accumulations of Δ^8,14- or Δ⁸-sterols and correspondingly large decreases in the ergosterol content are both implicated in the fungitoxicity of these compounds in N. haematococca. © 1998 Society of Chemical Industry     

Characterisation of energy-dependent efflux of imazalil and fenarimol in isolates of Penicillium italicum with a low,medium and high degree of resistance to DMI-fungicides

Differential accumulation of [¹⁴C]imazalil and [¹⁴C]fenarimol by germlings of wild-type and DMI-resistant isolates ofPenicillium italicum was studied at various pH values. At pH 7 and 8 the low-resistant isolate E_300–3 accumulated 22% and 35%, respectively, less imazalil than the wild-type isolate W₅. Imazalil accumulation at pH 5 and 6 was similar. Isolate E_300–3 also accumulated less fenarimol as compared with the wild-type isolate. This difference was much more obvious than for imazalil and was observed at all pH values tested. Differences in accumulation of both imazalil and fenarimol between low (E_300–3), medium (H₁₇) and high resistant (I₃₃) isolates were not observed. These results suggest that decreased accumulation of DMIs is responsible for a low level of resistance only and that additional mechanisms of resistance might operate in isolates with a medium and high degree of resistance. With all isolates fenarimol accumulation was energy-dependent. This was not obvious for imazalil.The wild-type and DMI-resistant isolates had a similar plasma membrane potential as determined with the probe [¹⁴C]tetraphenylphosphonium bromide ([¹⁴C]TPP⁺). Various test compounds, among which ATPase inhibitors, ionophoric antibiotics and calmodulin antagonists, affected the accumulation of [¹⁴C]TPP⁺, [¹⁴C]imazalil and [¹⁴C]fenarimol. No obvious correlation between the effects of the test compounds on accumulation levels of the fungicides and [¹⁴C]TPP⁺ could be observed. These results indicate that the plasma membrane potential does not mediate the efflux of DMI fungicides byP. italicum.     

Effects of imazalil on sterol composition of sensitive and DMI-resistant isolates of Penicillium italicum

Imazalil differentially inhibited dry weight increase of 10-hour-old germlings of wild-type and DMI-resistant isolates ofPenicillium italicum in liquid malt cultures. EC₅₀ values ranged from 0.005 to 0.27 μg ml^?1. In all isolates ergosterol constituted the major sterol (over 95% of total sterols) in the absence of the fungicide. Therefore, DMI-resistance cannot be associated to a deficiency of the C-14 demethylation enzyme in the ergosterol biosynthetic pathway. Imazalil treatment at concentrations around EC₅₀ values for inhibition of mycelial growth resulted in a decrease in ergosterol content and a simultaneous increase in 24-methylene-24,25-dihydrolanosterol content in all isolates. A correlation existed between the imazalil concentration necessary to induce such changes in sterol composition and the EC₅₀ values for inhibition of mycelial growth of the different isolates. The reason for the differential effects of imazalil on sterol composition in the variousP. italicum isolates may be due to decreased accumulation of the fungicide in the mycelium and to other yet non-identified mechanisms of resistance.     

Antifungal mode of action of triforine

Rapidly growing mycelia of Aspergillus fumigatus treated with 10 μg/ml triforine (N,N′-bis-(1-formamido-2,2,2-trichloroethyl)-piperazine) showed little or no inhibition in dry weight increase prior to 2 h. By 2.5–3 h, triforine inhibited dry weight increase by 85%. The effects of triforine on protein, DNA, and RNA syntheses corresponded to the effect on dry weight increase both in time of onset and magnitude. Neither glucose nor acetate oxidation were inhibited by triforine.Ergosterol synthesis was almost completely inhibited by triforine even in the first hour after treatment. Inhibition of ergosterol synthesis was accompanied by an accumulation of the ergosterol precursors 24-methylenedihydrolanosterol, obtusifoliol, and 14α-methyl-Δ^{8, 24 (28)}-ergostadienol. Mycelia treated with 5 μg/ml of triarimol (α-(2,4-dichlorophenyl)-α-phenyl-5-pyrimidinemethanol) also accumulated the same sterols as well as a fourth sterol believed to be Δ^{5, 7}-ergostadienol.Identification of 4,4-dimethyl-Δ^{8, 24 (28)}-ergostadienol in untreated mycelia indicates that the C-14 methyl group is the first methyl group removed in the biosynthesis of ergosterol by A. fumigatus. The lack of detectable quantities of 4,4-dimethyl-Δ^{8, 24 (28)}-ergostadienol in triforine or triarimol-treated mycelia and the accumulation of C-14 methylated sterols in treated mycelia suggests that both fungicides inhibit sterol C-14 demethylation. The accumulation of Δ^{5, 7}-ergostadienol in triarimol-treated mycelia further implies that triarimol also inhibits the introduction of the sterol C-22(23) double bond.Two strains of Cladosporium cucumerinum tolerant to triforine and triarimol were also tolerant to the fungicide S-1358 (N-3-pyridyl-S-n-butyl-S′-p-t-butylbenzyl imidodithiocarbonate).     

The effect of three different pesticides on soil protozoan activity

Three pesticides, dimethoate, pirimicarb and fenpropimorph, had a negative impact on the colonization of a sterilized soil by a natural population of soil protozoa. Fenpropimorph has an effect at the lowest concentration applied; 25 mg litre^?1. Dimethoate reduced the soil respiration significantly for about 10 days after application. In single species cultures of soil protozoa in liquid media, a flagellate Cercomonas sp., was a better test organism than a ciliate, Colpoda sp., or an amoeba, Acanthamoeba sp., with respect to interpretation of dose-response curves and ease of observation.     

Influence of soil pH and contents of organic carbon and clay on the volatilization of [14C]fenpropimorph after application to bare soil

The behaviour of the morpholine fungicide fenpropimorph applied to soil was investigated in a laboratory chamber. The volatility and metabolism of a ¹⁴C-labelled fenpropimorph formulation (Corbel^®) was studied after application to three soils (sandy loam, loamy clay and loamy sand), simulating a four-day weather scenario in the volatilization chamber. Additional experiments were conducted under standard climatic conditions over a period of 24 h using sandy soils with different pH values. The results of the first experiments showed that most of the radioactivity applied remained in the soils as unchanged fenpropimorph four days after application. In the experiments with the sandy loam and loamy clay, less than 5% of the applied radioactivity was removed by volatilization whereas 11·4% volatilized from the surface of the loamy sand. The comparatively higher volatilization of the fungicide from the loamy sand was confirmed by the later experiments indicating that higher soil pH favoured volatilization of [¹⁴C]fenpropimorph from sandy soils. Thus 5·6% (pH 5·0), 18·9% (pH 5·8) and 28·3% (pH 6·6) of the radioactivity applied volatilized within one day after application. The overall recoveries were between 93·8% and 111·3% in these experiments. © 1998 SCI     

Effect of propiconazole on growth and sterol biosynthesis by Sclerotium rolfsii

Germination of sclerotia ofSclerotium rolfsii on agar nutrient medium was delayed or slightly inhibited by concentrations of propiconazole between 0.4 and 4.0 μg ml^?1, but was strongly inhibited by 8 μg ml^?1 and completely inhibited by 16 μg ml^?1. On the other hand, growth of hyphae from the germinated sclerotia was strongly inhibited by propiconazole at 1 μg ml^?1 or greater. Hyphal growth from agar discs on agar medium was about 8 times less sensitive than hyphal growth from the sclerotia or from hyphal inoculum in liquid media. Propiconazole at 0.25 and 1.0 μg ml^?1 strongly inhibited ergosterol biosynthesis, but this was not associated with large accumulations of C-14 methyl sterols. The ratio of eburicol to ergosterol in hyphae grown in the presence of 0.25 μg ml^?1 propiconazole for 16, 30 or 45 h was 0.11, 0.13 and 0.04, respectively, for the three intervals while for hyphae grown in the presence of 1 μg ml^?1, the ratios were 0.29, 0.36 and 0.30, respectively, for the same intervals. In view of a ratio of 23.5 for¹⁴C-acetate incorporation into the two sterols during the initial 6 h growth period in the presence of propiconazole, it is believed that the lack of large accumulation of C-14 methyl sterols is due to the feedback inhibition by eburicol or to cell lysis when the content of ergosterol becomes too low in the actively growing cells.     

Mechanism of inhibition of sterol biosynthesis enzymes by N-substituted morpholines

Tridemorph, fenpropimorph and derivatives have been tested in vitro on Δ⁸→Δ⁷-sterol isomerase (SI) from maize seedlings. The results show that these N-substituted morpholines strongly inhibit this enzyme (I₅₀: 0.4 μM for fenpropimorph, 0.6 μM for tridemorph). In fenpropimorph, where a chiral centre is present at C-2 of the alkyl chain, good enantioselectivity was observed for enzyme inhibition. Inhibition of SI and of the cycloeucalenol-obtusifoliol isomerase (COI), another enzyme of plant sterol biosynthesis, is probably due to protonation of the N-substituted morpholines, giving a positively charged nitrogen atom, at the pH (7.4) of the enzyme assays. In order to test this hypothesis, the pH dependence of the inhibition constants has been measured. Fenpropimorph, (pK_a 7.5) which can be protonated to give a morpholinium cation and N-methylfenpropimorph, the corresponding quaternary ammonium derivative, have been tested as inhibitors in a pH range from 6 to 8.5. The I₅₀ value of fenpropimorph toward the SI increased 20 times as the pH increased from 6 to 8.5. In contrast the I₅₀ value of fenpropimorph for COI did not change significantly in this pH range. While the I₅₀ value of N-methyl fenpropimorph towards COI decreased more than 50 times as the pH increased from 6 to 8.5, its I₅₀ value for SI only varied slightly in this pH range. Taken together, these results suggest the existence of an interaction of the morpholinium cations with one or several enzyme amino acid residues of a much higher pK_a in the case of COI than SI. Moreover for maize COI, the pK_a of the amino acid residue(s) interacting with these morpholines derivatives is probably close to that of these molecules.     

Toxicity of fenpropimorph to fenarimol-resistant isolates of Penicillium italicum

Fenarimol-resistant isolates ofPenicillium italicum with cross-resistance to imidazole and triazole fungicides which inhibit ergosterol biosynthesis were tested for their sensitivity to fenpropimorph. Radial growth tests on PDA showed that the isolates (n=6) lacked cross-resistance to fenpropimorph or displayed enhanced sensitivity to the fungicide (negatively correlated cross-resistance). Control of blue mold decay of oranges incited by the wild-type isolate could be achieved by dipping fruits in suspensions of fenpropimorph at a concentration of 100 mg ml^–1. Decay of oranges incited by the fenarimol-resistant isolates was controlled at the same or at a lower concentration (30 mg ml^–1), thus showing that the normal or increased sensitivity to fenpropimorph is also expressed under in vivo conditions.Samenvatting Fenarimol-resistente isolaten vanPenicillium italicum met kruisresistentie tegen imidazool-en triazoolfungiciden die de ergosterolbiosynthese remmen, werden getoetst op hun gevoeligheid voor fenpropimorf. Radiale groeiproeven op PDA toonden aan dat de isolaten (n=6) geen kruisresistentie bezaten met fenpropimorf of een verhoogde gevoeligheid voor het middel vertoonden (negatief gecorreleerde kruisresistentie). Op sinaasappels konPenicillium-rot, veroorzaakt door het wild-type bestreden worden door middel van een dompelbehandeling met fenpropimorf bij een dosering van 100 g ml^–1). Bestrijding van rot veroorzaakt door fenarimil-resistente isolaten werd verkregen bij dezelfde of een lagere dosering (30 g ml^–1); aldus werd aangetoond dat de normale of verhoogde gevoeligheid voor fenpropimorf ook in vivo tot uiting komt.     

Sterol metabolism in wheat treated by N-substituted morpholines

Wheat caryopses were treated with racemic fenpropimorph. As shown previously in other plant species, 9(β,19-cyclopropyl sterols were found to accumulate markedly. A distinctive feature was a remarkable accumulation of 31 -norcyclobranol, a very rare sterol in nature. A⁸-sterols were also identified. The ratio A⁸-sterols: cyclopropyl sterols was shown to depend greatly on the configuration of the methyl substituent in position 2 of fenpropimorph. Whereas cyclopropyl sterols predominated in the case of seedlings treated with the (2S)-2-methyl enantiomer, A⁸-sterols were shown to be very abundant in plants treated with the (2R)-2-methyl enantiomer. A⁸-sterols were shown also to be more abundant in leaves than in roots. Experiments were conducted to find out whether the phytotoxic response of plants to fenpropimorph could be ascribed to its action on sterol biosynthesis. From the results obtained it appears that this is not the case and thus the phytotoxic effect is probably related to a cellular target other than sterol biosynthesis.     

Laboratory resistance to fungicides which inhibit ergosterol biosynthesis in Penicillium italicum

Laboratory isolates ofPenicillium italicum with varying levels of resistance to fenarimol were obtained via mass selection of conidia on fenarimol-amended PDA. All fenarimol-resistant isolates showed cross-resistance to other fungicides which inhibit ergosterol biosynthesis (bitertanol, etaconazole, fenapanil, and imazalil), but not to fenpropimorph. In contrast, all isolates with a relatively high degree of resistance to fenarimol, exhibited increased sensitivity to fenpropimorph (negatively correlated cross-resistance). The varying degrees of resistance to ergosterol biosynthesis inhibitors (EBI's) suggest that different mutations for resistance are involved. Isolates with a high degree of resistance were selected from conidial populations of isolates with a low resistance level. This indicates that in sich strains different mutations for resistance are present simultaneously.Somein vitro growth parameters of resistant isolates slightly differed from those of the wild type. Virulence of most resistant isolates on oranges was visually normal and in competition experiments with mixed inocula of wild-type and resistant isolates, the latter could still be isolated after five successive infection cycles on fungicide-free oranges. Nevertheless, the proportion of resistant conidia in the successive inocula gradually decreased.Decay of oranges inoculated with EBI-resistant isolates could still be controlled by a curative dip treatment with imazalil at dosage rates recommended in practice (500 g ml^–1). However, with the highly resistant isolates, decay control was not complete at half this dosage, indicating only a marginal control at the full dosage rate.On the basis of the results described it is assumed that at a high selection pressure of EBI's in practice, gradual accumulation of different mutations for resistance, together with selection of normal fitness may eventually lead to loss of control ofPenicillium decay. Therefore, desease control strategies with a low selection pressure of EBI's are advisable.Samenvatting Laboratoriumisolaten vanPenicillium italicum met uiteenlopende resistentieniveau's tegen fenarimol werden verkregen door massaselectie van conidiën op fenarimolbevattende PDA. Alle fenarimol-resistente isolaten vertoonden kruisresistentie tegen andere fungiciden die de ergosterolbiosynthese remmen (bitertanol, etaconazool, fenapanil, imazalil), maar niet tegen fenpropimorf. Alle isolaten met een relatief hoge graad van fenarimolresistentie waren zelfs gevoeliger voor dit laatste fungicide (negatief gecorreleerde kruisresistentie). De uiteenlopende graden van resistentie tegen ergosterolbiosynthese remmers (EBI's) suggereren dat verschillende mutaties een rol kunnen spelen. Isolaten met een hoge resistentiegraad werden geselecteerd in conidiënpopulaties van isolaten met een lage resistentiegraad. Dit duidt erop dat in dergelijke stammen verschillende mutaties gelijktijdig aanwezig zijn.Er werden kleine verschillen in parameters voorin vitro groei tussen resistente en gevoelige isolaten geconstateerd. De virulentie van vrijwel alle stammen op sinaasappels was normaal; in competitie-experimenten met mengpopulaties van gevoelige en resistente isolaten konden laatstgenoemde isolaten nog na vijf oppenvolgende infectiecycli op fungicide-vrije sinaasappels worden geïsoleerd. Desalniettemin nam het percentage resistente conidiën in de opeenvolgende inocula geleidelijk af. Penicillium-rot op sinaasappel, geïnoculeerd met EBI-resistente isolaten, kon nog worden bestreden door een curatieve dompelbehandeling met imazalil bij een dosering die in de praktijk wordt aanbevolen (500 g ml^–1). Bij een halvering van deze dosering werd echter op sinaasappels, geïnoculeerd met de hoog-resistente isolaten nog rot waargenomen, hetgeen erop duidt dat de bestrijding bij de volle dosering slechts marginaal is.Op grond van de beschreven resultaten kan worden verondersteld dat in de praktijk onder hoge selectiedruk van EBI's een geleidelijke accumulatie van verschillende mutaties, gepaard gaande met selectie van een normale fitheid, kan plaatsvinden, hetgeen uiteindelijk zou kunnen leiden tot onvoldoende bestrijding vanPenicillium-rot. Bestrijdingsstrategieën met een lage selectiedruk van EBI's zijn daarom wenselijk.     

Effect of the fungicide imazalil on hepatic microsomal cytochrome P-450 and cytochrome P-450 dependent activities in the Bobwhite quail (Colinus virginianus)

Bobwhite quails were treated with imazalil for 8 weeks. The fungicide was given admixed in the food at 0, 100, 300, 500 and 1000 mg kg^?1. Even at the highest dose tested, imazalil did not affect the liver weight or the hepatic microsomal protein content. In treated quails, no significant induction of cytochrome P-450 or NADPH-cytochrome c-reductase activity was observed. Furthermore, imazalil did not induce or inhibit 7-ethoxyresorufin or 7-ethoxycoumarin O-deethylase in quail microsomes. Only a slight but significant increase by 35% and 49% in aniline hydroxylase activity was measured for the 500- and 1000-mg kg^?1 dose levels, respectively. After a drug-free period of one week, aniline hydroxylase activity returned to control values, indicating that the effect was fully reversible. It is concluded that imazalil does not induce or inhibit drug-metabolizing enzymes in the quail, even at doses which exceed by far the maximum levels currently used to dress seed under field conditions (100 mg kg^?1).     

Effects of imazalil on sterol composition of sensitive and DMI-resistant isolates of Penicillium italicum

Imazalil differentially inhibited dry weight increase of 10-hour-old germlings of wild-type and DMI-resistant isolates ofPenicillium italicum in liquid malt cultures. EC₅₀ values ranged from 0.005 to 0.27 g ml^–1. In all isolates ergosterol constituted the major sterol (over 95% of total sterols) in the absence of the fungicide. Therefore, DMI-resistance cannot be associated to a deficiency of the C-14 demethylation enzyme in the ergosterol biosynthetic pathway. Imazalil treatment at concentrations around EC₅₀ values for inhibition of mycelial growth resulted in a decrease in ergosterol content and a simultaneous increase in 24-methylene-24,25-dihydrolanosterol content in all isolates. A correlation existed between the imazalil concentration necessary to induce such changes in sterol composition and the EC₅₀ values for inhibition of mycelial growth of the different isolates. The reason for the differential effects of imazalil on sterol composition in the variousP. italicum isolates may be due to decreased accumulation of the fungicide in the mycelium and to other yet non-identified mechanisms of resistance.Imazalil remt differentieel de toename in drooggewicht van 10-uur-oude gekiemde sporen van wild-type en DMI-resistente isolaten vanPenicillium italicum in vloeistofcultures van moutextract. De EC₅₀ waarden voor groei van de verschillende isolaten lopen uiteen van 0,005 tot 0,27 g ml^–1. In afwezigheid van het fungicide is in alle isolaten ergosterol het belangrijkste sterol (meer dan 95% van het totaal). DMI-resistentie kan daarom niet in verband staan met deficiëntie van het C-14 demethyleringsenzym in de ergosterol biosynthese. Imazalilbehandeling van mycelium bij concentraties rond de EC₅₀ waarde voor groeiremming, resulteerde bij alle isolaten in een afname van het ergosterolgehalte en een gelijktijdige toename van het gehalte aan 24-methyleen-24,25-dihydrolanosterol. Er bestaat dus een nauwe correlatie tussen de imazalilconcentratie die noodzakelijk is om vergelijkbare veranderingen in sterolsamenstelling te induceren en de EC₅₀ waarde voor remming van myceliumgroei van de verschillende isolaten. De differentiële effecten van imazalil op de sterolsamenstelling van de verschillendeP. italicum isolaten kunnen worden veroorzaakt door verminderde accumulatie van het fungicide in het mycelium en door andere, nog niet geïdentificeerde resistentiemechanismen.     

Novel inhibitors of sterol C-14 demethylase and Δ14 Reductase/Δ8 → Δ7 isomerase for cereal disease control

The activity of five experimental fungicides combining structural elements responsible for sterol C-14 demethylase inhibition and sterol nuclear double-bond transformations has been investigated by examining sterol accumulation in Ustilago maydis (DC.) Corda, inhibition of sterol-biosynthesis enzymes in Saccharomyces cerevisiae Meyer using a cell-free system and protective activity in cereals against Erysiphe graminis DC. and Puccinia coronata Corda. Combining the fenpropidin/fenpropimorph basic structure with a pyridine moiety offered no advantage in fungicidal activity. However, the combination of the pyrifenox structure with a morpholine moiety yielded a compound which was a good inhibitor of C-14 demethylase and Δ⁸ → Δ⁷ isomerase in vitro and gave good cereal protection.     

Fungitoxicity and growth regulation involving aspects of lipid biosynthesis

Triarimol and triforine inhibit ergosterol biosynthesis in fungi and cause accumulation of free fatty acids, 24-methylenedihydrolanosterol, obtusifoliol and 14α-methyl-δ^8,24(28)-ergostadienol. Triparanol also inhibits ergosterol synthesis and causes accumulation of free fatty acids, but not of the latter 3 sterols. Triparanol appears to inhibit prior to lanosterol in the sterol biosynthetic pathway of Ustilago maydis and at unidentified sites subsequent to lanosterol which lead to the accumulation of a sterol which migrates with desmethylsterols on TLC plates. Quantitative abnormalities in sterols and free fatty acids in U. maydis are not produced by the fungicides carbendazim, chloroneb, carboxin and cycloheximide. A deficiency in nitrogen leads to a marked increase in triglycerides, but a normal distribution pattern for other lipids.Inhibition of oxidative demethylation of the sterol 14α-methyl group is probably the prime mechanism of inhibition of ergosterol biosynthesis by triarimol. Rates of formation of obtusifoliol and 14α-methyl-δ^8,24(28)-ergostadienol in triarimol-treated U. maydis cells suggest that C-4 demethylation occurs along an abnormal pathway which operates effectively only at high substrate concentrations. The growth retardant action of triarimol and ancymidol in higher plants most likely results from inhibition of a reaction in the gibberellin biosynthetic pathway analogous to sterol C-14 demethylation.Free fatty acid accumulation in U. maydis cells treated with inhibitors of sterol synthesis are derived mainly from polar lipid degradation and from de novo synthesis as a consequence of the disproportionality between fatty acid synthesis and utilization. The free fatty acids may play a significant role in the lethality of these inhibitors in this organism.     

Chemistry and biology of novel amine fungicides: Attempts to improve the antifungal activity of fenpropimorph

In spite of considerable efforts by many workers, there has been a lack of progress in the area of amine fungicides since fenpropimorph. Random synthesis of a large variety of different amine compounds, as well as intelligent structural modification of the lead structure fenpropimorph (well over 15 000 amines have been screened at BASF alone) have not led to a new market product so far. Further work has been focused on the reported mode of action of fenpropimorph, notably on the inhibition of the sterol Δ¹⁴-reductase. Although some doubt has to be cast on the hypothesis that fenpropimorph behaves as a sterol mimic, the concept of ‘high energy intermediate’ inhibitors has been employed successfully. Rational drug design of azasterol mimics has led to a number of very potent inhibitors of the sterol Δ¹⁴-reductase which also displayed high fungicidal activity in the greenhouse. Although many of these compounds are more powerful reductase inhibitors than fenpropimorph, under field conditions none showed significant advantages over this established fungicide. Most likely, fenpropimorph already exhibits the maximum fungicidal activity which can be attained by blocking the sterol Δ¹⁴-reductase. This would mean that, with the development of the ‘second generation’ amine fungicide fenpropimorph, this class of compounds has already virtually been optimized.